Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes occurring in the female reproductive tract rendering the sperm able to fertilize constitute the phenomenon of "sperm capacitation". Using the mouse as an experimental model, we have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins (Visconti et al., 1995a; Visconti et al., 2002). We have also demonstrated that the increase in protein tyrosine phosphorylation as well as capacitation were regulated by a cAMP-dependent pathway involving protein kinase A (PKA) (Visconti et al., 1997; Visconti et al., 1995b). The presence of this regulatory pathway has subsequently been demonstrated in sperm from other species such as bovine (Galantino-Homer et al., 1997), human (Leclerc et al., 1996; Osheroff et al., 1999), boar (Kalab et al., 1998) and hamster (Visconti et al., 1999c). Despite these advances in understanding the mechanisms regulating phosphorylation during capacitation, little is known about the identity of the protein targets of this phosphorylation cascade and of the kinases and phosphatases involved in the regulation of capacitation. The objective of this proposal is to identify the proteins regulated by phosphorylation during capacitation. In addition, our efforts will be conducted to identify the kinase(s) involved in the regulation of capacitation of human and mouse sperm and to further elucidate the sequence of reactions that control protein tyr phosphorylation and capacitation. The hypotheses underlying this proposal postulate that proteins specifically phosphorylated during capacitation (as well as the kinases responsible for their phosphorylation) are involved in the regulation of the capacitation state of mammalian sperm. Characterization of the phosphorylated protein substrates and the kinase(s) involved in the regulation of capacitation will provide novel targets for pharmacological control of the fertilization process. To these ends, we propose the following aims: 1) To identify proteins which undergoes phosphorylation during sperm capacitation, 2) To identify and characterize kinases present in sperm playing a role in capacitation. 3) To investigate the regulation and function of sperm kinases and of proteins undergoing phosphorylation during capacitation. [unreadable] [unreadable]